Epithelial barrier loss is a pathogenic component of numerous diseases of the intestines and other organs. The multi-protein tight junction complex forms the paracellular barrier between adjacent cells and limits the flux of large and small solutes across the paracellular pathway. Many interactions between tight junction proteins have been described. The peripheral membrane scaffolding protein, zonula occludens-1 (ZO-1) mediates several of these interactions by direct binding through distinct domains. ZO-1 has been reported to interact with over 20 proteins, including claudins, ZO-2 and ZO-3, junctional adhesion molecule (JAM), G112, 1 catenin, occludin, F-actin, cortactin, and cingulin. Recent studies from our laboratory have shown that the tight junction is highly dynamic, even at steady state. Published studies and my preliminary data suggest that subtle changes in ZO-1 interactions can result in marked changes in the dynamic properties of tight junction proteins and barrier function. As further evidence that ZO-1 may be a critical determinant of barrier function, ZO-1 is inappropriately redistributed following treatment of epithelia with the cytokine TNF, which is central to barrier loss and pathogenesis in Crohn's and experimental inflammatory bowel disease. The precise mechanisms of ZO-1 involvement in barrier function are poorly understood, however. Thus, the objective of this proposal is to define the contributions of specific molecular interactions between ZO-1 and other tight junction proteins to ZO- 1 dynamic behavior, barrier function, and barrier regulation. Based on strong published and preliminary data, my central hypothesis is that interactions between ZO-1 and other proteins define normal trafficking and dynamic behaviors at steady state and in response to exogenous stimuli. To test this hypothesis, I will determine how specific ZO-1 domain deletions impact the trafficking and dynamic exchange of ZO-1 and other tight junction proteins to regulate epithelial barrier function (Aim 1) and define the roles of specific ZO-1 domains in response to physiologically- and pathophysiologically-relevant stimuli, including TNF (Aim 2). Within these aims, I will use innovative tools and techniques to comprehensively assess, for the first time, the impact of ZO-1 interactions on epithelial barrier structure and function. These studies are significant because the data, which are expected to identify ZO-1 domains that are linked to distinct disease-associated barrier defects, will benefit human health by providing information necessary to target specific ZO-1 interactions, restore barrier function, and prevent disease progression.